Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

Abstract

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

What is claimed is: 
     
       1. An engineered polypeptide comprising an amino acid sequence with at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8, wherein isoleucine at the position corresponding to residue 330 of SEQ ID NO: 8 is substituted with a different amino acid in the amino acid sequence of the engineered polypeptide, and wherein the engineered polypeptide has imine reductase activity. 
     
     
       2. The engineered polypeptide of  claim 1 , further comprising at least two additional substitutions at positions corresponding to SEQ ID NO: 8 selected from 9, 12, 31, 33, 37, 39, 43, 44, 46, 48, 55, 56, 65, 82, 93, 98, 108, 137, 138, 141, 142, 143, 153, 154, 155, 156, 159, 166, 167, 168, 173, 177, 178, 184, 185, 195, 197, 198, 199, 200, 201, 203, 205, 206, 210, 213, 215, 216, 218, 220, 221, 223, 224, 226, 234, 239, 242, 245, 251, 253, 256, 257, 259, 260, 261, 262, 263, 265, 266, 267, 268, 269, 272, 273, 274, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 289, 290, 291, 292, 293, 294, 341, and 349, wherein the polypeptide has imine reductase activity. 
     
     
       3. The engineered polypeptide of  claim 2 , wherein the amino acid sequence comprises at least one amino acid residue difference as compared to the amino acid sequence of SEQ ID NO:4, selected from 198, 153, 167, 265, 262, 108, 234, 284, 282, 220, 272, 256, 267, 242, 281, 197, 277, 224, and 143. 
     
     
       4. The engineered polypeptide of  claim 2 , wherein the amino acid sequence comprises at least one amino acid residue difference as compared to the amino acid sequence of SEQ ID NO:6, selected from: 283, 262, 9, 259, 220, 267, 153, 279, 200, 224, 256, 137, 143, 260, 261, 154, 276, and 185. 
     
     
       5. The engineered polypeptide of  claim 2 , wherein the amino acid sequence comprises at least one amino acid residue difference as compared to the amino acid sequence of SEQ ID NO:8, selected from:
 12, 31, 37, 44, 46, 48, 55, 56, 65, 82, 93, 98, 108, 137, 138, 141, 142, 143, 153, 154, 155, 156, 159, 166, 168, 173, 177, 178, 184, 185, 195, 197, 199, 200, 201, 203, 205, 206, 210, 213, 215, 216, 218, 220, 221, 223, 226, 234, 239, 242, 245, 251, 253, 256, 257, 259, 260, 261, 262, 263, 265, 266, 267, 268, 269, 273, 274, 277, 278, 279, 280, 281, 282, 283, 284, 285, 289, 290, 291, 292, 293, 294, and 349. 
 
     
     
       6. The engineered polypeptide of  claim 2 , wherein the imine reductase activity comprises converting under suitable reaction conditions ketone substrate compound 
       
         
           
           
               
               
           
         
       
       and amine substrate compound 
       
         
           
           
               
               
           
         
       
       to produce the amine product compound 
       
         
           
           
               
               
           
         
       
     
     
       7. The engineered polypeptide of  claim 6 , wherein the imine reductase activity in converting the ketone and amine substrate compound pair to the listed amine product compound under suitable reaction conditions is increased at least 2-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS:4, 6, 12, 30, 126, 173, 360, and/or 550. 
     
     
       8. The engineered polypeptide of  claim 1 , in which the amino acid sequence of the engineered polypeptide comprises a sequence selected from the group consisting of the even-numbered sequence identifiers of SEQ ID NOS:536, 902, 904, 924, 940, 946, 960, 974, 980, 984, 992, 998, 1010, 1014, 1020, 1028, 1044, 1048, 1050, 1072, 1092, 1096, 1118, 1124, 1140, 1142, 1188-1300.